Rotaviruses, newly classified members of the Reoviridae family, are an important cause of infantile diarrhea in a wide variety of mammalian species including man. Because they have a segmented genome, rotaviruses undergo genetic reassortment at high frequency during mixed infection. Rotavirus reassortants have been used to establish gene coding assignments for several major antigenic specificities and functional activities of these viruses. In addition, reassortants with characteristics that make them potential vaccine candidates have been isolated. We succeeded in isolating a large number of reassortant viruses following coinfection of cell cultures with a fastidious human rotavirus - D, DS-1, P, or ST3 (serotype 1, 2, 3, or 4) and a wild type bovine or rhesus rotavirus. These reassortants were specifically isolated as vaccine candidate strains. Wild type animal rotaviruses were used since these viruses are less likely to have silent point mutations than mutagenized ts virus. Analysis of the genotype of these reassortants indicates that many reassortants possess 10 genes of animal rotavirus origin and a single gene of human rotavirus origin. These single human rotavirus gene substitution reassortants have the serotype specificity of their human rotavirus parent as determined by plaque reduction neutralization assay. Such reassortants with a single human rotavirus gene substitution represent promising candidate live vaccine strains. Because of the narrow and highly restricted host range observed for rotaviruses recovered from a wide variety of species, it is likely that substitution of a large number of animal rotavirus genes for the corresponding genes of human rotavirus will lead to attenuation for humans. On the other hand the major protective antigen would be derived from the human rotavirus parent. These reassortants have been adapted to growth in DBS FRhL-2 cells. Successful passage in such a diploid cell strain further enhances their potential as vaccine candidate strains.